This invention relates to nucleic acid molecules, genes, and polypeptides that are related to microbial pathogenicity.
Pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. The expression of microbial pathogenicity is dependent upon complex genetic regulatory circuits. Knowledge of the themes in microbial pathogenicity is necessary for understanding pathogen virulence mechanisms and for the development of new xe2x80x9canti-virulence or anti-pathogenicxe2x80x9d agents, which are needed to combat infection and disease.
In one particular example, the opportunistic human pathogen, Pseudomonas aeruginosa, is a ubiquitous gram-negative bacterium isolated from soil, water, and plants (Palleroni, J. N. In: Bergey""s Manual of Systematic Bacteriology, ed., J. G. Holt, Williams and Wilkins, Baltimore, Md., pp. 141-172, 1984). A variety of P. aeruginosa virulence factors have been described and the majority of these, such as exotoxin A, elastase, and phospholipase C, were first detected biochemically on the basis of their cytotoxic activity (Fink, R. B., Pseudomonas aeruginosa the Opportunist: Pathogenesis and Disease, Boca Raton, CRC Press Inc., 1993). Subsequently, the genes corresponding to these factors or genes that regulate the expression of these factors were identified. In general, most pathogenicity-related genes in mammalian bacterial pathogens were first detected using a bio-assay. In contrast to mammalian pathogens, simple systematic genetic strategies have been routinely employed to identify pathogenicity-related genes in plant pathogens. Following random transposon-mediated mutagenesis, thousands of mutant clones of the phytopathogen are inoculated separately into individual plants to determine if they contain a mutation that affects the pathogenic interaction with the host (Boucher et al., J. Bacteriol. 168:5626-5623, 1987; Comai and Kosuge, J. Bacteriol. 149:40-46, 1982; Lindgren et al., J. Bacteriol. 168:512-522, 1986; Rahme et al., J. Bacteriol. 173:575-586, 1991; Willis et al., Mol. Plant-Microbe Interact. 3:149-156, 1990). Comparable experiments using whole-animal mammalian pathogenicity models are not feasible because of the vast numbers of animals that must be subjected to pathogenic attack.
We have identified and characterized a number of nucleic acid molecules, polypeptides, and small molecules (e.g., phenazines) that are involved in conferring pathogenicity and virulence to a pathogen. This discovery therefore provides a basis for drug-screening assays aimed at evaluating and identifying xe2x80x9canti-virulencexe2x80x9d agents which are capable of blocking pathogenicity and virulence of a pathogen, e.g., by selectively switching pathogen gene expression on or off, or which inactivate or inhibit the activity of a polypeptide which is involved in the pathogenicity of a microbe. Drugs that target these molecules are useful as such anti-virulence agents.
In one aspect, the invention features an isolated nucleic acid molecule including a sequence substantially identical to any one of BI48 (SEQ ID NO:1), ORF2 (SEQ ID NO:2), ORF3 (SEQ ID NO:4), ORF602c (SEQ ID NO:6), ORF214 (SEQ ID NO:8), ORF1242c (SEQ ID NO:10), ORF594 (SEQ ID NO:12), ORF1040 (SEQ ID NO:14), ORF1640c (SEQ ID NO:16), ORF2228c (SEQ ID NO:18), ORF2068c (SEQ ID NO:20), ORF1997 (SEQ ID NO:22), ORF2558c (SEQ ID NO:24), ORF2929c (SEQ ID NO:26), ORF3965c (SEQ ID NO:28), ORF3218 (SEQ ID NO:30), ORF3568 (SEQ ID NO:32), ORF4506c (SEQ ID NO:34), ORF3973 (SEQ ID NO:36), ORF4271 (SEQ ID NO:38), ORF4698 (SEQ ID NO:40), ORF5028 (SEQ ID NO:42), ORF5080 (SEQ ID NO:44), ORF6479c (SEQ ID NO:46), ORF5496 (SEQ ID NO:48), ORF5840 (SEQ ID NO:50), ORF5899 (SEQ ID NO:52), ORF6325 (SEQ ID NO:54), ORF7567c (SEQ ID NO:56), ORF7180 (SEQ ID NO:58), ORF7501 (SEQ ID NO:60), ORF7584 (SEQ ID NO:62), ORF8208c (SEQ ID NO:64), ORF8109 (SEQ ID NO:66), ORF9005Sc (SEQ ID NO:68), ORF8222 (SEQ ID NO:70), ORF8755c (SEQ ID NO:72), ORF9431c (SEQ ID NO:74), ORF9158 (SEQ ID NO:76), ORF10125c (SEQ ID NO:78), ORF9770 (SEQ ID NO:80), ORF9991 (SEQ ID NO82), ORF10765c (SEQ ID N0:84), ORF10475 (SEQ ID NO:86), ORF11095c (SEQ ID NO:88), ORF11264 (SEQ ID NO:90), ORF11738 (SEQ ID NO:92), ORF12348c (SEQ ID NO:94), ORF12314c (SEQ ID NO:96), ORF13156c (SEQ ID NO:98), ORF12795 (SEQ ID NO:100), ORF13755c (SEQ ID NO:210), ORF13795c (SEQ ID NO:212), ORF14727c (SEQ ID NO:214), ORF13779 (SEQ ID NO:216), ORF14293c (SEQ ID NO:218), ORF14155 (SEQ ID NO:220), ORF14360 (SEQ ID NO:222), ORF15342c (SEQ ID NO:224), ORF15260c (SEQ ID NO:226), ORF14991 (SEQ ID NO:228), ORF15590c (SEQ ID NO:230), ORF15675c (SEQ ID NO:232), ORF16405 (SEQ ID NO:234), ORF16925 (SEQ ID NO:236), ORF17793c (SEQ ID NO:238), ORF18548c (SEQ ID NO:240), ORF17875 (SEQ ID NO:242), ORF18479 (SEQ ID NO:244), ORF19027c (SEQ ID NO:246), ORF19305 (SEQ ID NO:248), ORF19519 (SEQ ID NO:250), ORF19544 (SEQ ID NO:252), ORF20008 (SEQ ID NO:254), ORF20623c (SEQ ID NO:256), ORF21210c (SEQ ID NO:258), ORF21493c (SEQ ID NO:260), ORF21333 (SEQ ID NO:262), ORF22074c (SEQ ID NO:264), ORF21421 (SEQ ID NO:266), ORF22608c (SEQ ID NO:268), ORF22626 (SEQ ID NO:270), ORF23228 (SEQ ID NO:272), ORF23367 (SEQ ID NO:274), ORF25103c (SEQ ID NO:276), ORF23556 (SEQ ID NO:278), ORF26191c (SEQ ID NO:280), ORF23751 (SEQ ID NO:282), ORF24222 (SEQ ID NO:284), ORF24368 (SEQ ID NO:286), ORF24888c (SEQ ID NO:288), ORF25398c (SEQ ID NO:290), ORF25892c (SEQ ID NO:292), ORF25110 (SEQ ID NO:294), ORF25510 (SEQ ID NO:296), ORF26762c (SEQ ID NO:298), ORF26257 (SEQ ID NO:300), ORF26844c (SEQ ID NO:302), ORF26486 (SEQ ID NO:304), ORF26857c (SEQ ID NO:306), ORF27314c (SEQ ID NO:308), ORF27730c (SEQ ID NO:310), ORF26983 (SEQ ID NO:312), ORF28068c (SEQ ID NO:314), ORF27522 (SEQ ID NO:316), ORF28033c (SEQ ID NO:318), ORF29701c (SEQ ID NO:320), ORF28118 (SEQ ID NO:322), ORF28129 (SEQ ID NO:324), ORF29709c (SEQ ID NO:326), ORF29189 (SEQ ID NO:328), ORF29382 (SEQ ID NO:330), ORF30590c (SEQ ID NO:332), ORF29729 (SEQ ID NO:334), ORF30221 (SEQ ID NO:336), ORF30736c (SEQ ID NO:338), ORF30539 (SEQ ID NO:340), ORF31247c (SEQ ID NO:342), ORF39063c (SEQ ID NO:344), ORF31539c (SEQ ID NO:346), ORF31222 (SEQ ID NO:348), ORF31266 (SEQ ID NO:350), ORF31661c (SEQ ID NO:352), ORF32061c (SEQ ID NO:354), ORF32072c (SEQ ID NO:356), ORF31784 (SEQ ID NO:358), ORF32568c (SEQ ID NO:360), ORF33157c (SEQ ID NO:362), ORF32530 (SEQ ID NO:364), ORF33705c (SEQ ID NO:366), ORF32832 (SEQ ID NO:368), ORF33547c (SEQ ID NO:370), ORF33205 (SEQ ID NO:372), ORF33512 (SEQ ID NO:374), ORF33771 (SEQ ID NO:376), ORF34385c (SEQ ID NO:378), ORF33988 (SEQ ID NO:380), ORF34274 (SEQ ID NO:382), ORF34726c (SEQ ID NO:384), ORF34916 (SEQ ID NO:386), ORF35464c (SEQ ID NO:388), ORF35289 (SEQ ID NO:390), ORF35410 (SEQ ID NO:392), ORF35907c (SEQ ID NO:394),ORF35534 (SEQ ID NO:396), ORF35930 (SEQ ID NO:398), ORF36246 (SEQ ID NO:400), ORF26640c (SEQ ID NO:402), ORF36769 (SEQ ID NO:404), ORF37932c (SEQ ID NO:406), ORF38640c (SEQ ID NO:408), ORF39309c (SEQ ID NO:410), ORF38768 (SEQ ID NO:412), ORF40047c (SEQ ID NO:414), ORF40560c (SEQ ID NO:416), ORF40238 (SEQ ID NO:418), ORF40329 (SEQ ID NO:420), QRF40709c (SEQ ID NO:422), ORF40507 (SEQ ID NO:424), ORF41275c (SEQ ID NO:426), ORF42234c (SEQ ID NO:428), ORF41764c (SEQ ID NO:430), ORF41284 (SEQ ID NO:432), ORF41598 (SEQ ID NO:434), ORF42172c (SEQ ID NO:436), ORF42233c (SEQ ID NO:451), 33A9 (SEQ ID NO:102, 189, 190, 191, 192, 193, 194, 195, 196, 197, and 198), 34B12 (SEQ ID NO:104), 34B12-ORF1 (SEQ ID NO:105), 34B12-ORF2 SEQ ID NO:106), 36A4 (SEQ ID NO:109), 36A4 contig (SEQ ID NO:111), 23A2 (SEQ ID NO:112), 3E8 phn(xe2x88x92)(SEQ ID NO:114), 3E8 contigPAO1 (SEQ ID NO:115), 34H4 (SEQ ID NO:118), 33C7 (SEQ ID NO:119), 25a12.3 (SEQ ID NO:120), 8C12 (SEQ ID NO:121), 2A8 (SEQ ID NO:122), 41A5 (SEQ ID NO:123), 50E12 (SEQ ID NO:124), 35A9 (SEQ ID NO:125), pho23 (SEQ ID NO:126), 16G12 (SEQ ID NO:127), 25F1 (SEQ ID NO:128), PA14 degP (SEQ ID NO:131), 1126 contig (SEQ ID NO:135), contig 1344 (SEQ ID NO:136), ORFA (SEQ ID NO:153), ORFB (SEQ ID NO:154), ORFC (SEQ ID NO:155), phzR (SEQ ID NO:164, and 1G2 (SEQ ID NO:137). Preferably, the isolated nucleic acid molecule includes any of the above-described sequences or a fragment thereof; and is derived from a pathogen (e.g., from a bacterial pathogen such as Pseudomonas aeruginosa). Additionally, the invention includes a vector and a cell, each of which includes at least one of the isolated nucleic acid molecules of the invention; and a method of producing a recombinant polypeptide involving providing a cell transformed with a nucleic acid molecule of the invention positioned for expression in the cell, culturing the transformed cell under conditions for expressing the nucleic acid molecule, and isolating a recombinant polypeptide. The invention further features recombinant polypeptides produced by such expression of an isolated nucleic acid molecule of the invention, and substantially pure antibodies that specifically recognize and bind such a recombinant polypeptides.
In an another aspect, the invention features a substantially pure polypeptide including an amino acid sequence that is substantially identical to the amino acid sequence of any one of ORF2 (SEQ ID NO:3), ORF3 (SEQ ID NO:5), ORF602c (SEQ ID NO:7), ORF214 (SEQ ID NO:9), ORF1242c (SEQ ID NO:11), ORF594 (SEQ ID NO:13), ORF1040 (SEQ ID NO:15), ORF1640c (SEQ ID NO:17), ORF2228c (SEQ ID NO:19), ORF2068c (SEQ ID NO:21), ORF1997 (SEQ ID NO:23), ORF2558c (SEQ ID NO:25), ORF2929c (SEQ ID NO:27), ORF3965c (SEQ ID NO:29), ORF3218 (SEQ ID NO:31), ORF3568 (SEQ ID NO:33), ORF4506c (SEQ ID NO:35), ORF3973 (SEQ ID NO:37), ORF4271 (SEQ ID NO:39), ORF4698 (SEQ ID NO:41), ORF5028 (SEQ ID NO:43), ORF5080 (SEQ ID NO:45), ORF6479c (SEQ ID NO:47), ORF5496 (SEQ ID NO:49), ORF5840 (SEQ ID NO:51), ORF5899 (SEQ ID NO:53), ORF6325 (SEQ ID NO:55), ORF7567c (SEQ ID NO:57), ORF7180 (SEQ ID NO:59), ORF7501 (SEQ ID NO:61), ORF7584 (SEQ ID NO:63), ORF8208c (SEQ ID NO:65), ORF8109 (SEQ ID NO:67), ORF9005c (SEQ ID NO:69), ORF8222 (SEQ ID NO:71), ORF8755c (SEQ ID NO:73), ORF9431c (SEQ ID NO:75), ORF9158 (SEQ ID NO:77), ORF10125c (SEQ ID NO:79), ORF9770 (SEQ ID NO:81), ORF9991 (SEQ ID NO:83), ORF10765c (SEQ ID NO:85), ORF10475 (SEQ ID NO:87), ORF11095c (SEQ ID NO:89), ORF11264 (SEQ ID NO:91), ORF11738 (SEQ ID NO:93), ORF12348c (SEQ ID NO:95), ORF12314c (SEQ ID NO:97), ORF13156c (SEQ ID NO:99), ORF12795 (SEQ ID NO:101), ORF13755c (SEQ ID NO:211), ORF13795c (SEQ ID NO:213), ORF14727c (SEQ ID NO:215), ORF13779 (SEQ ID NO:217), ORF14293c (SEQ ID NO:219), ORF14155 (SEQ ID NO:221), ORF14360 (SEQ ID NO:223), ORF15342c (SEQ ID NO:225), ORF15260c (SEQ ID NO:227), ORF14991 (SEQ ID NO:229), ORF15590c (SEQ ID NO:231), ORF15675c (SEQ ID NO:233), ORF16405 (SEQ ID NO:235), ORF16925 (SEQ ID NO:237), ORF17793c (SEQ ID NO:239), ORF18548c (SEQ ID NO:241), ORF17875 (SEQ ID NO:243), ORF18479 (SEQ ID NO:245), ORF19027c (SEQ ID NO:247), ORF19305 (SEQ ID NO:249), ORF19519 (SEQ ID NO:251), ORF19544 (SEQ ID NO:253), ORF20008 (SEQ ID NO:255), ORF20623c (SEQ ID NO:257), ORF21210c (SEQ ID NO:259), ORF21493c (SEQ ID NO:261), ORF21333 (SEQ ID NO:263), ORF22074c (SEQ ID NO:265), ORF21421 (SEQ ID NO:267), ORF22608c (SEQ ID NO:269), ORF22626 (SEQ ID NO:271), ORF23228 (SEQ ID NO:273), ORF23367 (SEQ ID NO:275), ORF25103c (SEQ ID NO:277), ORF23556 (SEQ ID NO:279), ORF26191c (SEQ ID NO:281), ORF23751 (SEQ ID NO:283), ORF24222 (SEQ ID NO:285), ORF24368 (SEQ ID NO:287), ORF24888c (SEQ ID NO:289), ORF25398c (SEQ ID NO:291), ORF25892c (SEQ ID NO:293), ORF25110 (SEQ ID NO:295), ORF25510 (SEQ ID NO:297), ORF26762c (SEQ ID NO:299), ORF26257 (SEQ ID NO:301), ORF26844c (SEQ ID NO:303), ORF26486 (SEQ ID NO:305), ORF26857c (SEQ ID NO:307), ORF27314c (SEQ ID NO:309), ORF27730c (SEQ ID NO:311), ORF26983 (SEQ ID NO:313), ORF28068c (SEQ ID NO:315), ORF27522 (SEQ ID NO:317), ORF28033c (SEQ ID NO:319), ORF29701c (SEQ ID NO:321), ORF28118 (SEQ ID NO:323), ORF28129 (SEQ ID NO:325), ORF29709c (SEQ ID NO:327), ORF29189 (SEQ ID NO:329), ORF29382 (SEQ ID NO:331), ORF30590c (SEQ ID NO:333), ORF29729 (SEQ ID NO:335), ORF30221 (SEQ ID NO:337), ORF30736c (SEQ ID NO:339), ORF30539 (SEQ ID NO:341), ORF31247c (SEQ ID NO:343), ORF30963c (SEQ ID NO:345), ORF31539c (SEQ ID NO:347), ORF31222 (SEQ ID NO:349), ORF31266 (SEQ ID NO:351), ORF31661c (SEQ ID NO:353), ORF32061c (SEQ ID NO:355), ORF32072c (SEQ ID NO:357), ORF31784 (SEQ ID NO:359), ORF32568c (SEQ ID NO:361), ORF33157c (SEQ ID NO:363), ORF32530 (SEQ ID NO:365), ORF33705c (SEQ ID NO:367), ORF32832 (SEQ ID NO:369), ORF33547c (SEQ ID NO:371), ORF33205 (SEQ ID NO:373), ORF33512 (SEQ ID NO:375), ORF33771 (SEQ ID NO:377), ORF34385c (SEQ ID NO:379), ORF33988 (SEQ ID NO:381), ORF34274 (SEQ ID NO:383), ORF34726c (SEQ ID NO:385), ORF34916 (SEQ ID NO:387), ORF35464c (SEQ ID NO:389), ORF35289 (SEQ ID NO:391), ORF35410 (SEQ ID NO:393), ORF35907c (SEQ ID NO:395), ORF35534 (SEQ ID NO:397), ORF35930 (SEQ ID NO:399), ORF36246 (SEQ ID NO:401), ORF26640c (SEQ ID NO:403), ORF36769 (SEQ ID NO:405), ORF37932c (SEQ ID NO:407), ORF38640c (SEQ ID NO:409), ORF39309c (SEQ ID NO:41 1), ORF38768 (SEQ ID NO:413), ORF40047c (SEQ ID NO:415), ORF40560c (SEQ ID NO:417), ORF40238 (SEQ ID NO:419), ORF40329 (SEQ ID NO:421), ORF40709c (SEQ ID NO:423), ORF40507 (SEQ ID NO:425), ORF41275c (SEQ ID NO:427), ORF42234c (SEQ ID NO:429), ORF41764c (SEQ ID NO:431), ORF41284 (SEQ ID NO:433), ORF41598 (SEQ ID NO:435), ORF42172c (SEQ ID NO:437), ORF42233c (SEQ ID NO:152), 33A9 (SEQ ID NOS:103, 199, 200, 201, 202, 203, 204, 205, 206, 207, and 208), 34B12-ORFJ (SEQ ID NO:107), 34B12-ORF2 (SEQ ID NO:108), 36A4 (SEQ ID NO:110), 3E8phzA (SEQ ID NO:116), 3E8phzB (SEQ ID NO:117), PhzR (SEQ ID NO:165), ORFA (SEQ ID NO:156), ORFB (SEQ ID NO:157), ORFC (SEQ ID NO:158), and PA14 degP (SEQ ID NO:132). Preferably, the substantially pure polypeptide includes any of the above-described sequences of a fragment thereof; and is derived from a pathogen (e.g., from a bacterial pathogen such as Pseudomonas aeruginosa).
In yet another related aspect, the invention features a method for identifying a compound which is capable of decreasing the expression of a pathogenic virulence factor (e.g., at the transcriptional or post-transcriptional levels), involving (a) providing a pathogenic cell expressing any one of the isolated nucleic acid molecules of the invention; and (b) contacting the pathogenic cell with a candidate compound, a decrease in expression of the nucleic acid molecule following contact with the candidate compound identifying a compound which decreases the expression of a pathogenic virulence factor. In preferred embodiments, the pathogenic cell infects a mammal (e.g., a human) or a plant.
In yet another related aspect, the invention features a method for identifying a compound which binds a polypeptide, involving (a) contacting a candidate compound with a substantially pure polypeptide including any one of the amino acid sequences of the invention under conditions that allow binding; and (b) detecting binding of the candidate compound to the polypeptide.
In addition, the invention features a method of treating a pathogenic infection in a mammal, involving (a) identifying a mammal having a pathogenic infection; and (b) administering to the mammal a therapeutically effective amount of a composition which inhibits the expression or activity of a polypeptide encoded by any one of the nucleic acid molecules of the invention. In preferred embodiments, the pathogen is Pseudomonas aeruginosa. 
In yet another aspect, the invention features a method of treating a pathogenic infection in a mammal, involving (a) identifying a mammal having a pathogenic infection; and (b) administering to the mammal a therapeutically effective amount of a composition which binds and inhibits a polypeptide encoded by any one of the amino acid sequences of the invention. In preferred embodiments, the pathogenic infection is caused by Pseudomonas aeruginosa. 
Moreover, the invention features a method of identifying a compound which inhibits the virulence of a Pseudomonas cell, involving (a) providing a Pseudomonas cell; (b) contacting the cell with a candidate compound; and (c) detecting the presence of a phenazine, wherein a decrease in the phenazine relative to an untreated control culture is an indication that the compound inhibits the virulence of the Pseudomonas cell. In preferred embodiments, the cell is Pseudomonas aeruginosa; the cell is present in a cell culture; and the phenazine is detected by spectroscopy (e.g., pyocyanin is detected at an absorbance of 520 nm). Pyocyanin is generally detected according to any standard method, e.g., those described herein.
By xe2x80x9cisolated nucleic acid moleculexe2x80x9d is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide of the invention that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. A substantially pure polypeptide of the invention may be obtained, for example, by extraction from a natural source (for example, a pathogen); by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid molecule exhibiting at least 25% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 30%, 40%, 50%, 60%, more preferably 80%, and most preferably 90% or even 95% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between exe2x80x943 and exe2x80x94100 indicating a closely related sequence.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a polypeptide of the invention.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule).
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody. A purified antibody of the invention may be obtained, for example, by affinity chromatography using a recombinantly-produced polypeptide of the invention and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant a compound or antibody which recognizes and binds a polypeptide of the invention but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
By xe2x80x9cderived fromxe2x80x9d is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, or combination thereof).
By xe2x80x9cinhibiting a pathogenxe2x80x9d is meant the ability of a candidate compound to decrease, suppress, attenuate, diminish, or arrest the development or progression of a pathogen-mediated disease or an infection in a eukaryotic host organism. Preferably, such inhibition decreases pathogenicity by at least 5%, more preferably by at least 25%, and most preferably by at least 50%, as compared to symptoms in the absence of the candidate compound in any appropriate pathogenicity assay (for example, those assays described herein). In one particular example, inhibition may be measured by monitoring pathogenic symptoms in a host organism exposed to a candidate compound or extract, a decrease in the level of symptoms relative to the level of pathogenic symptoms in a host organism not exposed to the compound indicating compound-mediated inhibition of the pathogen.
By xe2x80x9cpathogenic virulence factorxe2x80x9d is meant a cellular component (e.g., a protein such as a transcription factor, as well as the gene which encodes such a protein) without which the pathogen is incapable of causing disease or infection in a eukaryotic host organism.
The invention provides a number of targets that are useful for the development of drugs that specifically block the pathogenicity of a microbe. In addition, the methods of the invention provide a facile means to identify compounds that are safe for use in eukaryotic host organisms (i.e., compounds which do not adversely affect the normal development and physiology of the organism), and efficacious against pathogenic microbes (i.e., by suppressing the virulence of a pathogen). In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for an anti-virulence effect with high-volume throughput, high sensitivity, and low complexity.
The methods are also relatively inexpensive to perform and enable the analysis of small quantities of active substances found in either purified or crude extract form.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.